Capillary electrophoresis ("CE") is a known chemical sample separation technique that is of increasing interest to those concerned with analyzing the separation of the chemical contents of a chemical sample. It is a modification of electrophoresis, and is typically practiced in a thin glass capillary instead of on a 2-dimensional surface such as paper or in a gel. This technique offers the benefits of high efficiency and resolution, rapid separations, the ability to analyze small sample amounts, and a desirable simplicity from the point of view of the apparatus required when compared to competing analytical techniques such as gel electrophoresis, gas chromatography, and liquid chromatography. As in all separation systems, high resolution, sensitivity and dynamic range are the end objectives.
The benefits of capillary electrophoresis derive to a large extent from the use of narrow diameter capillary tubes, which permit efficient removal of the heat generated in the separation process. This heat removal prevents convective mixing which would degrade the separating power. The narrow diameter tubes also allow high voltages to be used to generate the electric field in the capillary while limiting current flow and hence heat generation.
A CE separation begins by filling the capillary with a supporting electrolyte. Next, a small amount of sample is injected into one end of the capillary. Typical sample injection volumes range from 1-20 nanoliters. After sample injection, and with each end of the capillary immersed in a buffer solution, a high voltage is applied to the capillary and the sample components are separated on the basis of different ion mobility. A capillary electrophoretic separation can also be augmented with a bulk fluid flow, called electro-osmotic flow. If present, it adds a constant-velocity component to all species in the capillary.
In a CE separation implementing absorption techniques, the contents of the capillary tube are then analyzed by passing light of a certain wavelength through the capillary tube and then detecting the amount of light which has passed through the tube using a photodetector. The wavelength used is chosen to coincide with an absorption band of the sample components of interest, usually in the ultraviolet or visible regions of the spectrum. The photodetector output is digitally processed and used to compute the absorbance of the sample. An electropherogram, i.e. the plot of absorbance versus time, is typically stored in a computer file and may be displayed on, for example, a PC screen.
With conventional systems if a plurality of capillary tubes are to be used, a corresponding number of light sources, photodetectors, amplifiers and A/D converters must also be used. The outputs of the plurality of A/D converters are fed into signal processing circuitry such as a Digital Signal Processing chip to time average the data, compress it and send it to a personal computer for storage and/or display.
It has become very practical and desirable to use a plurality of capillary tubes in parallel, since this enables more samples to be analyzed at the same time. However, using the conventional systems described above results in a very expensive and large-sized assembly since much duplication of circuitry is necessary. For each capillary tube in the parallel configuration, the apparatus requires a corresponding D.C. light source, photodetector, amplifier and A/D converter. Such redundancy of components disadvantageously impacts, among other things, the cost and size of such parallel analytical instruments.
An example of a known multiple capillary electrophoresis separation system using absorbance detection is disclosed in European Patent Application 0 581 413 A2. In the disclosed apparatus, light from a light source is directed to the measurement region of each capillary, using a plurality of optical fibers. A second set of optical fibers routes transmitted light to a plurality of photo detectors, each of which has its own amplifier and signal processing electronics. The optical fiber configuration results in complexity, light loss and reduced analytical sensitivity. Additionally, as discussed, such an implementation disadvantageously has numerous redundant components.
Another example of multiple capillary electrophoresis is provided in U.S. Pat. No. 5,324,401 which employs fluorescence detection. In this invention, a continuous source of light from a coherent light source, a laser, is delivered to a plurality of optical fibers, each one delivering excitation light to a respective separation capillary. The measurement regions of the capillaries are arranged adjacent to each other such that emitted fluorescent light can be imaged using a CCD camera. It should be noted that lasers do not generally have the required stability for absorbance measurements, are much more expensive, and less suitable for use in routine analytical apparatus. In addition, suitable lasers do not exist with wavelengths near the absorption maximum of many analytes. Furthermore, a CCD camera generally is not a suitable choice as a detector for an absorbance measurement because for absorbance, light levels are typically high and lead to saturation of the CCD pixels before they can be read out. Reducing the light source output to suit the CCD detector results in reduced analytical sensitivity and dynamic range. Fluorescence measurements, in contrast, are low light level measurements, well matched to the characteristics of a CCD detector.
Another class of multiple capillary separations are referenced and their limitations critiqued in U.S. Pat. No. 5,324,401. The referenced systems use detection implementations where the measurement region of each capillary is scanned sequentially. Typically, a confocal method is used to sequentially measure laser excited fluorescence from an array of separation capillaries. The numerous disadvantages of such systems include, as applied to absorption measurements as discussed hereinbefore, the limitations of a laser beam as a light source, and relatively greater expense associated with complex optics.